1. Field of the Invention
The present invention relates to novel biologically active polypeptides, more particularly, artificially created polypeptides which are commonly capable of inducing the production of interferon-gamma (hereinafter abbreviated as xe2x80x9cIFN-xcex3xe2x80x9d) by immunocompetent cells.
2. Description of the Prior Art
The present inventors successfully isolated some polypeptides which are capable of inducing the production of IFN-xcex3 by immunocompetent cells, as well as cloned cDNAs which encode the polypeptides (hereinafter called xe2x80x9cthe wild-type polypeptidesxe2x80x9d); the relating inventions are disclosed in Japanese Patent Kokai Nos.27,189/96 and 193,098/96 and Japanese Patent Application No.67,434/96. It is known that the wild-type polypeptides usually contain SEQ ID NOs:1-3 as consensus partial amino acid sequences, and that they usually possess properties of inducing the formation of killer cells and enhancing their cytotoxicities, in addition to the property of inducing production of IFN-xcex3, a useful biologically active protein. Because of the properties, the use of the wild-type polypeptides as antiviral, antimicrobial, antitumor, and/or anti-immunopathic agents, etc. is in great expectation.
However, as described in Japanese Patent Application No.67,434/96 by the present applicant, there is the problem that natural cells commonly do not enable producing the wild-type polypeptide in desired amounts. The present inventors energetically investigated the cause, revealing that the wild-type polypeptides usually exist in the form of precursor exhibiting no biological activity in natural cells. The precursor has been proved to be converted into an active form by the action of converting enzymes such as interleukin-1xcex2 converting enzymes, which is described in Japanese Patent Application Nos.207,691/96 and 213,267/96 by the present applicant.
The wild-type polypeptides are probably instable, which would be involved in the above problem as another cause. Progress in recombinant DNA techniques of recent years have facilitated to remove and/or replace one or more amino acids in biologically active proteins to develop mutagenized polypeptides. However, even the progressed recombinant DNA techniques couldn""t improve the stability of every protein with the inherent activity, unless taking trails and errors on the proteins individually.
In view of the foregoing, the first object of the present invention is to provide a polypeptide with significantly improved stability, while substantially retaining a biological activity of the wild-type polypeptide.
The second object of the present invention is to provide a process for producing the polypeptide.
The third object of the present invention is to provide a use of the polypeptide for a pharmaceuticals.
The present inventors energetically studied to attain the above objects, revealing that a polypeptide is more stable than the wild-type polypeptide, wherein the stale polypeptide contain an amino acid sequence derived either from a polypeptide containing the partial amino acid sequences of SEQ ID NOs:1-3 by replacing one or more of the cysteines with a different amino acid(s), or from the cysteine-replaced amino acid sequences by adding, removing and/or replacing one or more amino acids to and/or at position(s) excepting the position(s) where the cysteine(s) has been replaced; and that some of the stable polypeptides, in which the cysteine(s) have been replaced, exhibit an activity of inducing the production of IFN-xcex3 by immunocompetent cells significantly higher than the wild-type polypeptides. These polypeptides proved to be easily produced in a desired amount by using recombinant DNA techniques and to exhibit less toxicities. Based on the above, the present polypeptides were confirmed to be effectively used not only as an IFN-xcex3 inducer but also as a pharmaceutical.
The first object of the present invention is attainable by an isolated polypeptide which is capable of inducing the production of interferon-gamma by immunocompetent cells, said polypeptide containing either amino acid sequence wherein one or more cysteines are replaced with different amino acid(s) while leaving respective consensus sequences as shown in SEQ ID NOs:1-3 intact, or that wherein one or more amino acids are added, removed and/or replaced at one or more sites including those in the consensus sequences but excluding those of the replaced cysteine.
The second object of the present invention is attainable by a process for producing a polypeptide, which comprises the steps of culturing a cell containing a DNA encoding the present polypeptide to produce a polypeptide, and collecting the produced polypeptide from the resulting culture.
The third object of the present invention is attainable by an agent for susceptive diseases, which contains the present polypeptide as an effective ingredient.